During the past year we have continued to pursue our long-term goal of elucidating the mechanisms by which proteins are targeted to and translocated across biological membranes. Currently we are studying protein secretion in pathogenic bacteria via the autotransporter or type V pathway. Autotransporters are very large (~100-400 kD) virulence factors that contain a small C-terminal ?b domain? that facilitates translocation of a large N-terminal ?passenger domain? across the outer membrane (OM) by an unknown mechanism. Following their translocation across the OM, many passenger domains are released from the cell surface by proteolytic cleavage. We have been using an autotransporter produced by E. coli O157:H7 called EspP as a model protein in our experiments. Like ~10% of the autotransporters identified to date, EspP contains an exceptionally long signal peptide that is distinguished by a conserved N-terminal extension. We have found that the N-terminal extension promotes posttranslational translocation by a novel mechanism which involves altering the accessibility of the signal peptide to targeting factors and the protein translocation machinery in the inner membrane (the SecYEG complex). We have also been studying the mechanism by which the EspP passenger domain is translocated across the OM. Using Blue Native PAGE, analytical ultracentrifugation and other biochemical methods we have found that the EspP b domain behaves as a compact monomer and forms a channel that is too narrow to accommodate folded polypeptides. Surprisingly, we found that a folded protein domain attached to the N-terminus of EspP is efficiently translocated across the OM and that the native EspP passenger domain folds at least partially in the periplasm. These apparently paradoxical data strongly suggest that an external factor transports the passenger domain across the OM and that the b domain functions primarily as a membrane anchor. Our results challenge the prevailing view of the autotransporter b domain as a protein translocase and provide new insights into the biogenesis of a large class of proteins that play important roles in bacterial pathogenesis. Very recently, we have also obtained insights into the mechanism by which the EspP passenger domain is cleaved from the cell surface.[unreadable] [unreadable] In separate studies we have been investigating the regulation of the expression of SecA, a cytoplasmic ATPase that plays a major role in the translocation of proteins through the SecYEG complex. The gene that encodes SecA resides just downstream of the gene that encodes SecM, and the two genes form a single operon. When secretion is impaired, a 17 amino acid motif near the C-terminus of SecM induces a translation arrest from within the ribosome tunnel. As a consequence, the structure of the mRNA is altered and SecA is translated more efficiently. We have used a novel application of fluorescence resonance energy transfer (FRET) to gain insight into the mechanism of SecM translation arrest. We found that the SecM C-terminus adopts a compact conformation upon synthesis of the arrest motif. This conformational change does not occur spontaneously, but rather is induced by the ribosome. Translation arrest requires both compaction of the SecM C-terminus and the presence of key residues in the arrest motif. Further analysis showed that the arrested peptidyl-tRNA was resistant to puromycin treatment and revealed additional changes in the ribosome-nascent SecM complex. Based on these results, we have proposed that translation arrest results from a series of reciprocal interactions between the ribosome and the C-terminus of the nascent SecM polypeptide.